In eukaryotic cells, the SV40-based shuttle vector, pZ189, has been utilized in vivo and in vitro, to analyze mutations in response to UV and benzpyrene (BP). These two agents yield different frequencies and specificities of mutations in vivo. Moreover, mutation fixation (with similar spectra as in vivo) occurs during replication of UV and BP-damaged pZ189 in vitro (using cell extracts plus SV40 large T antigen to promote DNA replication). In a gap-filling mutagenesis assay, purified polymerase alpha (HeLa cell) is highly inaccurate during DNA synthesis, but accuracy is improved several-fold if HeLa single-stranded DNA binding protein (SSB) is also present. Two BP enantiomers, (+) and (-)anti, form adducts with DNA, but the (+) form is far more carcinogenic. When pZI89 was adducted with the two enantiomers, and the vector "shuttled" in vivo, the (+) form had a greater effect on the replication complex both in terms of lesion bypass and nucleotide misincorporation. In the in vitro replication system, the (+) form again had a greater effect. Studies in E.coli have focused on the UmuD cleavage reaction, which is inefficient in vitro but is required for mutagenesis in vivo. We now find, using an ultrasensitive chemiluminescent assay, that UmuD cleavage is also inefficient in vivo. To elucidate the role of the vital but inefficiently produced cleavage product, UmuD', we have over-produced UmuD' protein by placing an engineered UmuD' gene under the control of the strong lambda PL promoter. Unlike E.coli, S.typhimurium is poorly mutable. However, we found that the Salmonella UmuD protein is cleaved to UmuD', indicating that the defect in mutagenesis is not due to a defect in processing. When mammalian cells are pretreated with a carcinogen (UV or mitomycin C), the repair of UV-damaged DNA (in the form of a transfected vector) is enhanced. We have identified a constitutive DNA damage-specific, DNA binding (DDB) protein complex which is induced to high levels by this pretreatment. DDB protein is the first such damage-specific, damage-inducible DNA binding protein identified in primate cells. DDB protein is not detectable nor inducible in patients with the repair deficient, cancer-prone disease, xeroderma pigmentosum. Thus, DDB may play a central role in DNA repair. With regard to DNA replication, our focus is on a viral gene product, SV40 small t antigen (tag). This protein is required for the efficient induction of cellular DNA replication and transformation when non-dividing cells are infected in vitro by SV40, while large Tag alone suffices in proliferating cells. We found that SV40 tag deletion mutants readily transform rapidly proliferating cell types (e.g., lymphoblast, osteoblast) in vivo, leading to lymphomas and osteosarcomas in infected hamsters; animals infected with wild type SV40 develop tumors of nonproliferating cells (i.e., fibrosarcomas). SV40 tag may effect a change in the phosphorylation state of Tag, which may in turn be necessary for Tag-induced DNA replication.